The intracellular distribution of regulatory molecules may provide a mechanism for controlling gene expression. The subcellular location of cAMP-dependent protein kinase was analyzed in living cells by microinjection of regulatory and catalytic subunits labeled with fluorescein. Following microinjection, type I holoenzyme was found in the cytoplasm and remained there for up to 4 hr. Upon dissociation of holoenzyme with 8-bromo-cAMP, free catalytic subunit appeared in the nucleus while regulatory subunit remained in the cytoplasm. Similarly, purified catalytic subunit was transported to the nucleus in the absence of elevated intracellular cAMP following its introduction into the cytoplasm. Translocation to the nucleus was apparent within 10 min and persisted for at least 2 hr. In contrast, purified regulatory subunit, like holoenzyme, was maintained in the cytoplasm. These results suggest that one function of the type I regulatory subunit is to serve as a cytoplasmic anchor, sequestering the catalytic subunit in the cytoplasm until holoenzyme dissociates in response to increased cAMP.